This invention relates to genetically engineered endothelial cells and, in particular, to endothelial cells which have been modified to resist lysis and activation by complement and evade the host's immune mechanisms for removing foreign cells, when inserted into a non-autologous host.
Endothelial cells are specialized cells which form the lining of the heart and the blood vessels. Because of their direct contact with the circulating blood, a number of proposals have been made to genetically engineer these cells and use them as “in vivo” drug delivery systems, for example, by Culliton, B. J. 1989. “Designing Cells to Deliver Drugs,” Science 246:746-751; and Zwiebel et al., “High-Level Recombinant Gene Expression in Rabbit Endothelial Cells Transduced by Retroviral Vectors,” Science 243:220-222 (transfer of a human adenosine deaminase gene and a rat growth hormone gene to aortic endothelial cells using a retroviral vector and demonstration of the secretion of rat growth hormone from such cells after seeding onto a synthetic vascular graft).
Natural endothelial cells play important roles in normal physiology. In particular, these cells constitute the interface between the blood and the vessel wall and the organs of the body. As such, endothelial cells secrete various natural products directly into the blood stream, maintain an antithrombotic surface on the inside of the vessel, restrict leukocytes from penetrating the vessel wall, regulate various of the biological properties of smooth muscle cells, and participate in the control of vessel wall tone. Therefore, loss of endothelial cells results in the loss of these normal physiological processes and ultimately leads to pathological conditions such as coronary artery disease, organ transplant rejection and vasculitis.
Accordingly, in addition to their use as a medium for the in vivo administration of therapeutics, there is a need to provide genetically engineered endothelial cells to replace natural endothelial cells which have been lost due to disease or surgery.
In the past, proposals and/or efforts to use endothelial cells for either administration of therapeutics or cell replacement have generally been limited to autologous cells, i.e., cells derived from the organism undergoing treatment. Alternatively, the patient must be immunosuppressed, which is costly and leaves the patient vulnerable to infection.
This approach has suffered from a number of problems. For example, it is difficult to harvest healthy endothelial cells from the individual to be treated in significant quantities. The procedures for doing so require removal of a section of vasculature and then scraping or otherwise dislodging the endothelial cells from the walls of the vessels. As a result, to be useful for cell replacement, a large number of autologous endothelial cells must be grown in culture. To be of practical use, especially in the case of cell replacement, this culturing must take place quickly. Unfortunately, the cell doubling time for endothelial cells is on the order of at least 24 to 48 hours, leading to time periods on the order of a week or more before sufficient quantities of endothelial cells are available for genetic engineering or cell replacement. In addition, under normal physiological conditions, the cell doubling time for natural endothelial cells in vivo is also prolonged, making naturally occurring cell replacement in vivo following endothelial cell loss or damage highly inefficient.
Although the use of autologous endothelial cells for cell replacement therapy appears to be a difficult task, the potential use of microvascular capillary endothelial cells for systemic delivery of a therapeutic protein may allow for the use of either autologous or immunoprotected allogeneic or xenogeneic capillary endothelial cells. Several investigators have proposed the use of implanted genetically modified fibroblasts or keratinocytes as delivery systems and, while plausible, the expressed protein must diffuse through interstitial tissues, and into the microcirculation in order to gain access to the vascular system. This diffusional barrier to the systemic circulation is a considerable impediment to achieving adequate plasma levels of the desired therapeutic protein. Endothelial cells offer several advantages over fibroblasts in that they secrete their protein products directly into the bloodstream. Further, fibroblasts via their excessive production of fibrotic scar tissue, can prove highly detrimental to the host.
When a foreign cell is transplanted into a host, the immune system of the host rapidly mobilizes to destroy the cell and thereby protect the host. The immune system attack on the foreign cell is referred to as transplant rejection. The organism's first line of defense is through either lytic destruction or the activation of procoagulant and prothrombotic properties of the donor endothelial cell that may result from activation of the host's complement system and is generally known as the “hyperacute rejection response” or simply the “hyperacute response.”
Several studies have demonstrated that the hyperacute response to transplants of either xenogeneic (from different species) and allotypic (from different individuals of the same species) organs is mediated by antibody-dependent activation of the complement system at the surface of the donor endothelium, as discussed, for example, by Platt et al. , 1990 “Transplantation of discordant xenografts: a review of progress” Immunology Today 11:450-456. That is, the 0complement_system_attacks the endothelial cells lining the vessels of the transplanted organ. In studies of in vivo animal model systems aimed at assessing hyperacute rejection, 15 minutes after revascularization, xenoreactive antibodies were found to be deposited on the surface of the donor endothelium (Pober et al., Human Immunol.28, 258-262 (1990); Haisch et al., Surgery 108, 306-311 (1990); Vercellotti et al., J. Immunol.146, 730-734 (1991)). This was followed by activation of the complement system. Following complement activation there was significant aggregation and adhesion In of platelets to the endothelium as well as formation of microthrombi and the migration of neutrophils and granulocytes into the interstitium. Ultimately, endothelial cells were destroyed, resulting in tissue ischemia and necrosis.
In addition to the rejection problems with xenografts, ten percent of allogeneic solid donor organs in HLA-identical matches have been found to be rejected by antibody/complement-mediated mechanisms (Brasile et al., Trans, Proceed. 
19, 894-895 (1987)). In 78% of the cases of rejection under these conditions, the antibodies are directed against the vascular endothelial cells (Brasile et al., Trans. 40, 672-675 (1985)).
The results of these xenograft and allograft transplantations demonstrate that complement activation on the surface of the endothelial cell plays an important and early role in the process of graft rejection.
The complement system is a complex interaction of plasma proteins and membrane cofactors which act in a multistep, multi-protein cascade sequence in conjunction with other immunological systems of the host organism. The classic complement pathway involves an initial antibody recognition of, and binding to, an antigenic site on a target cell. This surface bound antibody subsequently reacts with the first component of complement, Clq, forming a Cl-antibody complex with Ca2+, Clr, and Cls which is proteolytically active. Cls cleaves C2 and C4 into active components, C2a and C4a. The C4b,2a complex is an active protease called C3 convertase, and acts to cleave C3 into C3a and C3b. C3b forms a complex with C4b,2a to produce C4b,2a,3b, which cleaves C5 into C5a and C5b. C5b forms a complex with C6 and this complex interacts with C7 in the fluid phase thereby exposing hydrophobic domains within C5b and C6 that stabilize the C5b,6,7 ternary complex in the cell membrane. C8, which is in the fluid phase, then binds to the C5b, 6, 7 ternary complex and this complex may contribute to the development of functional membrane lesions and slow cell lysis. Upon binding of C9 to C8 in the C5b-8 membrane complex, lysis of foreign cells is rapidly accelerated.
Control of the complement system is necessary in order to prevent destruction of autologous cells. One of the central molecules in the complement cascade is C3b which aggregates in increasing amounts on foreign substances or organisms thereby targeting them for removal. The complement precursor proteins are activated to form C3b in either of two ways: (i) by interacting with antibody bound to a foreign target (classical pathway) or (ii) non-specifically by progressive and rapidly increasing accumulation on foreign substances on the surface of foreign cells (the alternative pathway).
Activation of the alternative pathway relies on molecular structures on the target cell to upset the delicate balance of the proteins involved so that their activation and deposition are focused on the surface of the target cell. In the alternative pathway C3b is continuously activated at a slow rate in the fluid phase by various agents including endotoxin, lipopplysaccharide, and serum proteases that convert C3 to C3b. C5b can also be formed from C5 by plasmin, elastase and other serum proteases to initiate formation of the MAC.
In order to control this process of complement activation and to protect normal syngeneic cells from indiscriminate destruction, a family of cell-surface proteins has evolved that interacts with C3b molecules. These proteins are as follows:
(a) Membrane cofactor protein (MCP or CD46) which exists on all cells, except red blood cells, and binds to C3b and activates molecules that cleave C3b into inactive fragments before it can accumulate on the surface of a target cell to destroy that cell.
(b) Decay accelerating factor (DAF or CD55) which exists on all cells including red blood cells and prevents C3b from reacting with other complement components preventing destruction of the cell. CD55, unlike CD46, does not destroy C3b.
(c) Complement receptor 1 (CR1 or CD35) which exists on a select group of lymphocytes as well as erythrocytes, neutrophils, and eosinophils and causes degradation of C3b molecules adhering to neighboring cells.
(d) Factor H and C4b-binding protein which both inhibit C3 convertase activity of the alternative complement pathway.
All of these proteins are encoded at a single chromosomal location (chromosome 1, band 1q32) identified as the RCA, i.e., the regulators of complement activation. They are each uniquely characterized structurally by a short consensus repeating unit (SCR) of approximately 60 amino acids composed mostly of cysteine, proline, glycine, tryptophan, and several hydrophobic residues. Reid, et al., Immunol. Today 7, 230 (1986); Coyne, et al., J. Immunol.149, 2906-2913 (1992). For CD46 and CD55, these SCRs are known to encode the functional domains of the proteins necessary for full complement regulatory activity. Adams, et al., J. Immunol.147, 3005-3011 (1991). For a discussion of SCRs generally see Perkins et al., Biochem. 27, 4004-4012 (1988); for a discussion of SCRs of factor H and CD35 see Krych et al., Proc. Natl. Acad. Sci. USA 88, 4353-4357 (1991) and Weisman et al., Science 249, 146 (1990).
In addition to membrane and soluble inhibitors of the C3 convertase enzymes, human blood cells and the vascular endothelium express a cell surface glycoprotein, CD59, that serves to prevent assembly of the C5b-9 lytic MAC and, therefore, protects these cells from complement-mediated cell activation and lysis. U.S. Pat. No. 5,135,916 issued Aug. 4, 1992, assigned to the Oklahoma Medical Research Foundation, and U.S. Ser. No. 07/729,926 filed Jul. 15, 1991, assigned to the Oklahoma Medical Research Foundation and Yale University, disclose that the human complement regulatory protein CD59 can be used to protect non-human endothelial cells, for example, porcine endothelial cells, from attack by human complement, either when provided in solution with the cells or expressed in genetically engineered cells. See also Zhao et al., 1991 “Amplified gene expression in CD59-transfected Chinese Hamster Ovary cells confers protection against the membrane attack complex of human complement” J. Biol. Chem. 266:13418-13422. The homologous complement inhibitory activity of CD59 resides in its species-specific interaction with the terminal complement components C8 and C9, as further reported by Rollins and Sims, 1990 “The complement inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9” J. Immunol.144:3478-3483.
Although the use of CD59 does successfully address the problem of hyperacute rejection as a result of complement attack, it does not protect the cell against the overall immune attack of the host organism against foreign endothelial cells.
In stimulating immune responses, antigens elicit many molecular and cellular changes. Lymphocytes recognize antigens as foreign and are responsible for initiating both cellular and humoral responses against the presenting antigen. B lymphocyte cells respond to antigen by the production of antibodies against the presenting antigen; T lymphocytes respond by initiating a cellular response to the presenting antigen. The two major subsets of T cells are TH cells, involved in processing of antigen for presentation to B cells, characterized by the presence of a cell-surface glycoprotein called CD4, and cytolytic T lymphocytes (CTLs), involved in recognition of antigen on cell surfaces and lysis of cells recognized as foreign, characterized by the presence of a cell-surface glycoprotein called CD8. T cells recognize peptide fragments in conjunction with one of the two main classes of cell-surface glycoproteins of the major histocompatibility complex (MHC): either class I (MHC-I) or class II (MHC-II) proteins. CD8+ T cells recognize antigens in conjunction with MHC-I, whereas CD4+ T cells recognize them in conjunction with MHC-II.
The MHC contain three major classes of genes. Class I genes encode the principal subunits of MHC-I glycoproteins, called human leukocyte antigens in humans, the principle ones being HLA-A, B, and C. These are present on virtually all cells and play a major role in rejection of allografts. They also form complexes with peptide fragments of viral antigens on virus-infected cells: recognition of the complexes by CD8+ CTLs results in destruction of virus infected cells. Recognition of the complexes is by a single receptor on the T cells which recognizes antigen in combination with MHC.
Class II genes, the major classes in humans being known as DP, DQ (subclasses β2, α2, and β1 α1) and DR (subclasses β1, β2, β3 and α1), encode cell-surface glycoproteins that are expressed by antigen-presenting cells, principally B cells, macrophages and dendritic cells. Together with peptide fragments of antigens, the class II proteins form the epitopes that are recognized by helper cells (CD4+). Class III genes encode at least three proteins of the complement cascade and two cytotoxic proteins, tissue necrosis factor and lymphotoxin, which are involved in diverse immune reactions that destroy cells.
T-cell mediated immune reactions can be organized into three sequential activation steps. First, CD4+ and CD8+ Tlymphocytes (T-cells) recognize the presence of non-autologous MHC class II and class I proteins, respectively, on the surface of the foreign cell.
Second, the T-cells are activated by interaction of a ligand with the T cell receptors and other accessory stimulatory molecules, so that activation depends upon a variety of variables including humoral signals such as cytokines received by protein receptors on the surface of the cells. Most important is the interaction between the antigen specific T cell receptor and ligand, a complex of MHC and antigenic peptide on the antigen presenting cell (APC). Other receptors present on the T cell must also be contacted by their ligands on APC to insure activation. Once activated, the T-cells synthesize and secrete interleukin-2 (IL-2) and other cytokines.
The cytokines secreted by the activated T-cells lead to the third, or effector, phase of the immune response which involves recruitment and activation of lymphocytes, monocytes, and other leukocytes which together lead to cell lysis, as reviewed, for example, by Pober et al., 1990 “The potential roles of vascular endothelium in immune reactions” Human Immunol.28:258-262.
Historically, attempts to interrupt the T-cell immune response have generally met with limited success. For example, several strategies have tried to use reagents of various types, including antibodies and blocking proteins, to interfere with adhesion between T-cells and foreign cells. Lider et al., 1988 “Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis” Science 239:181 reported on the use of T-cell vaccines; Owhashiand et al., 1988 “Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors specific for myelin basic protein” J. Exp. Med.168:2153, reported on the use of T-cell receptor blocking antibodies; Brostoffand et al. 1984 “Experimental allergic encephalomyelitis: successful treatment in vivo with a monoclonal antibody that recognizes T helper cells” J. Immunol.133:1938 reported on the use of antibodies to CD4; and Adorini et al., 1988 “Dissociation of phosphoinositide hydrolysis and Ca2+ fluxes from the biological responses of a T-cell hybridoma” Nature 334:623-628, reported on the use of blocking peptides that occupy T-cell receptors. These strategies have generally resulted in immune responses to the reagents, rather than the desired interruption of T-cell binding.
It would clearly be advantageous if one could decrease the probability of T-cell mediated reaction against transplanted cells, as well as complement-mediated attack and lysis of the cell.
It is therefore an object of the present invention to provide an improved method and compositions for constructing endothelial cells that are resistant to both complement and cellular attack when transplanted into a foreign host.
It is a further object of the present invention to provide genetically engineered cells that are not recognized as foreign when implanted into a foreign host and therefore evade attack by the immune system.
It is still further object of this invention to provide genetically engineered cells which after transplantation can resist complement-mediated attack and evade lymphocyte-mediated lysis, specifically CD4+T-lymphocytes, and preferably CD8+ T-lymphocytes.
It is another object of the invention to provide a mechanism for selectively killing such genetically engineered cells when their presence in the host is no longer desired.
It is still another object of the present invention to provide a biological vehicle for delivery of therapeutic products.